The objective of the proposed research is to optimize a safe and effective multi-antigen vaccine for smallpox using a single-cycle, replication-incompetent, alphavirus replicon vaccine vector that has been derived from attenuated strains of Venezuelan equine encephalitis (VEE). Preliminary studies have identified four candidate vaccinia virus (VACV) genes A33R, B5R, A27L and L1R (4pox) that when expressed in animals, elicit a protective immune response. Complete protection from intranasal challenge with VACV (strain IHD- J) could be demonstrated after as few as one 4pox virus-like replicon particle (VRP) immunization in a mouse model. The specific aims of this proposal are (1) to optimize the immunizing dose of individual VRP components of the 4pox vaccine, (2) to perform scale-up development of processes for manufacture of VRP vaccines expressing VACV genes, (3) to manufacture a 4pox VRP vaccine suitable for clinical evaluation, and (4) to perform toxicology tests of the 4pox VRP vaccine. The certified eradication of smallpox followed by the presumed destruction of all but two of the world's stocks of variola virus, led to the cessation of most smallpox vaccination programs. As a result, most of the world's population today has little or no immunity to smallpox. Orthopoxviruses remain a concern for a number of reasons. Naturally occurring orthopoxvirus disease and the threat of Orthopoxviruses used as biological weapons are just two examples. Much of the threat posed by Orthopoxviruses could be eliminated by vaccination;however, because the smallpox vaccine is a live orthopoxvirus the vaccine itself can pose serious health risks to immunocompetent individuals and especially to immunocompromised individuals. Based on the proposed studies, a safe and effective alternative smallpox vaccine will be carried through preclinical studies, pilot lot production, manufacture of clinical material and toxicology testing suitable to support submission of an IND (IND submission is not part of the scope of this proposal).